Research and diagnostic procedures require rapid, accurate and qualitative and/or quantitative determinations of substances (“analytes”) that are present in biological samples, such as biological tissues or fluids, at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, dental plaque, gingival crevicular fluid, and other biological specimens is desirably determined in an accurate and rapid fashion for suitable diagnosis or treatment.
In many cases, an analyte is identified in a sample using a compound that specifically recognizes the chemical features of the analyte. Often, monoclonal antibodies specific for one or more chemical epitopes on an analyte are used. The complex formed between the antibody and analyte can be detected by a variety of known methods. The most commonly used methods employ a signal generating moiety of some type which is either already attached to the antibody, or becomes attached to the antibody through further reaction. For example, in the formation of a complex of biotin with avidin, the complex may be detected using a label on either the avidin or biotin molecule. Such a label can be a radioisotope or an enzyme conjugated with the avidin or biotin. Alternatively, the avidin-biotin complex might be detected by further reaction with a labeled molecule which is specific to either or both parts of the complex. It is commonly known to do the same with antigens and their corresponding antibodies.
In diagnostic tests designed to be rapid and easy to use with moderate training in a doctor's office or clinic, the specific binding ligand of interest (such as an antigen from an infectious agent) is often detected using colorimetric, fluorescent or chemiluminescent signals resulting from reaction of the enzyme label with its corresponding substrate.
There is a need to produce the signal quickly and intensely if the ligand is present. This is commonly done using a colorimetric detection reagent. Upon addition of the detection reagent a colored product is produced. In many types of assay, the generation of color is not limited. In order to optimally quantitate the result, a stop reagent is employed to stop the formation of color and hold it at a stable level to allow for accurate quantitation. Acids such as sulfuric acid and hydrochloric acid can be used to stop the production of detectable signal when peroxidase is used as a label in specific binding reactions. However, the use of these acids has problems associated with it, namely corrosivity, short signal duration, and toxicity.
The investigator has discovered there is a need to provide a non-corrosive stop reagent and a stop reagent that allows for an extended dynamic range and a longer, more stable signal time than previous acids have provided.